Small molecule bcl-x1/bcl-2 binding inhibitors

ABSTRACT

Bcl-xL/Bcl-2 binding inhibitors useful in the treatment of unwanted proliferating cells, including cancers and precancers, in subjects in need of such treatment. Also provided are methods of treating a subject having unwanted proliferating cells comprising administering a therapeutically effective amount of a Bcl-xL/Bcl-2 binding inhibitor described herein to a subject in need of such treatment. Also provided are methods of preventing the proliferation of unwanted proliferating cells, such as cancers and precancers, in a subject comprising the step of administering a therapeutically effective amount of a Bcl-xL/Bcl-2 binding inhibitor described herein to a subject at risk of developing a condition characterized by unwanted proliferating cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/638,519 filed Dec. 22, 2004, the entirety of which isincorporated herein by reference.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

The present invention was funded, at least in part, by NationalInstitutes of Health Grant CA-94829. The government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

Thiazolidenediones (TZDs), including troglitazone (TG), rosiglitazone(RG), pioglitazone (PG), and ciglitazone (CG), are synthetic ligands ofthe peroxisome proliferator-activated receptor γ (PPARγ) (1). Thisfamily of PPARγ agonists improves insulin sensitivity by increasingtranscription of certain insulin-sensitive genes involved in themetabolism and transport of lipids, thus representing a new class oforal antidiabetic agents. More recently, certain TZDs, especially TG andCG, have also been shown to inhibit the proliferation of many cancercell lines that express high levels of PPARγ, including, but not limitedto, those of colon, prostate, breast, and liposarcoma [review: (2)]. AsPPARγ-mediated effects of TZDs promote the differentiation ofpreadipocytes, one school of thought attributes the same mechanism tothe terminal differentiation and cell cycle arrest of tumor cells (3).However, the PPARγ-activated target genes that mediate theantiproliferative effects remain elusive, as genomic responses to PPARγactivation in cancer cells are highly complicated (4). Reported causalmechanisms include attenuated expression of protein phosphatase 2A (5),cyclins D1 and E, inflammatory cytokines and transcription factors (2),and increased expression of an array of gene products linked to growthregulation and cell maturation (4). On the other hand, several lines ofevidence indicate that the inhibitory effect of TZDs on tumor cellproliferation was independent of PPARγ activation. For example, theantitumor effects appear to be structure-specific irrespective ofpotency in PPARγ activation, i.e., TG and CG are active while RG and PGare not. Also, there exists a three-orders-of-magnitude discrepancybetween the concentration required to produce antitumor effects and thatto mediate PPARγ activation. To date, an array of non-PPARγ targets havebeen implicated in the antitumor activities of TG and/or CG in differentcell systems, which include intracellular Ca²⁺ stores (6),phosphorylating activation of ERKs (extracellular signal-regulatedkinases) (7, 8), JNK (c-Jun N-terminal protein kinase), and p38 (9),up-regulation of early growth response-1 (10), p27^(kip1) (11),p21^(WAF/cIP1) (12), p53, and Gadd45 (13), and altered expression ofBcl-2 family members (9). However, some of these targets appear to becell type-specific due to differences in signaling pathways regulatingcell growth and survival in different cell systems.

In light of the potential use of TZDs in prostate cancerprevention/treatment (14, 15), signaling mechanisms whereby these PPARγagonists inhibit the proliferation of prostate cancer cells representthe focus of this investigation. We report here the development of novelTZD derivatives that lack activity in PPARγ activation but retain theability to induce apoptosis in two prostate cancer cell lines withdistinct PPARγ expression status, suggesting that these twopharmacological activities are unrelated. More importantly, wedemonstrate that TZD-mediated apoptosis was attributable, in part, tothe inhibition of the anti-apoptotic functions of Bcl-xL and Bcl-2 bydisrupting the BH3 domain-mediated interactions with pro-apoptotic Bcl-2members. From a translational perspective, dissociation of these twopharmacological activities, i.e., PPARγ activation and Bcl-xL/Bcl-2inhibition, provides a molecular basis to use Δ2-TG as a scaffold togenerate a novel class of Bcl-xL/Bcl-2 inhibitors. Accordingly, wedeveloped a structurally optimized Δ2-TG derivative (TG-88) with high invivo potency in inhibiting PC-3 tumor growth.

SUMMARY OF THE INVENTION

Provided are Bcl-xL/Bcl-2 binding inhibitors useful in the treatment ofunwanted proliferating cells, including cancers and precancers, insubjects in need of such treatment. Also provided are methods oftreating a subject having unwanted proliferating cells comprisingadministering a therapeutically effective amount of a compound describedherein to a subject in need of such treatment. Also provided are methodsof preventing the proliferation of unwanted proliferating cells, such ascancers and precancers, in a subject comprising the step ofadministering a therapeutically effective amount of a compound describedherein to a subject at risk of developing a condition characterized byunwanted proliferating cells. In some embodiments, the compoundsdescribed herein reduce the proliferation of unwanted cells by inducingapoptosis in those cells. In one embodiment, the compounds describedherein are used in the treatment of prostate cancer in a subject in needof such treatment. In another embodiment, the compounds described hereinare used in a method of preventing prostate cancer in a subject, whereinthe subject is at risk of developing prostate cancer. In someembodiments, the methods treating unwanted proliferating cells,including cancers and precancers, comprise inducing apoptosis in theunwanted proliferating cells by administering an effective amount of theBcl-xL/Bcl-2 binding inhibitor to the subject in need of such treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Development of PPARγ-inactive TZD derivatives. (A) Chemicalstructures of TG, RG, PG, CG, and the respective Δ2-derivatives. (B)Δ-2-TZD derivatives lack activity in PPARγ activation. Analysis of PPARγactivation was carried out as described in the Materials and Methods. Inbrief, PC-3 cells were exposed to individual test agents (10 μM) or DMSOvehicle in 10% FBS-supplemented RPMI 1640 medium for 48 h. Amounts ofactivated PPARγ in the resulting nuclear extracts were analyzed by PPARγtranscription factor ELISA kit. Each data point represents mean±S.D.(n=3). *P<0.01.

FIG. 2 Evidence that the effect of TG on apoptosis in prostate cancercells is dissociated from PPARγ activation. (A) Relative PPARγ levels inPC-3 and LNCaP cells, normalized to α-tubulin levels. Each data pointrepresents mean±S.D. (n=3). Inset: Western blot analysis of theexpression status of PPARγ (z,999 and α-tubulin (lower lane) in PC-3 andLNCaP cells. *P<0.01. (B) Dose-dependent effects of TG and Δ2-TG on thecell viability of PC-3 and LNCaP cells. PC-3 cells were exposed to TG orΔ2-TG at the indicated concentrations in serum-free RPMI 1640 medium in96-well plates for 24 h, and cell viability was assessed by MTT assay.Each data point represents the mean±S.D. (n=6). (C) Evidence ofapoptotic death in drug-treated PC-3 cells. Left panel, levels ofcytochrome c release into cytoplasm induced by different doses of TG andΔ2-TG. Values are means±S.D. (n=3), normalized to β-actin levels.*P<0.01. PC-3 cells were treated with either agent at the indicatedconcentration for 24 h in serum-free RMPI 1640 medium for 24 h, andmitochondria-free lysates were prepared. Equivalent amounts of proteinfrom individual lysates were electrophoresed, and probed by Westernblotting with anti-cytochrome c antibody (inset; β-actin blots are notshown). Right panel, formation of nucleosomal DNA in PC-3 cells thatwere treated with TG or Δ2-TG at the indicated concentrations for 24 h.DNA fragmentation was quantitatively measured by a cell death detectionELISA kit. Each data point represents mean±S.D. (n=3).

FIG. 3 Differential effects of CG, RG, PG, and their Δ2-derivatives onapoptotic death in PC-3 cells. (A) Dose-dependent effects of CG andΔ2-CG on PC-3 cell viability (left panel) and mitochondrial cytochrome crelease (right panel). Values are means±S.D. (n=3). Cytochrome c releaselevels are normalized β-actin levels. *P<0.01. (B) Dose-dependenteffects of RG and Δ2-RG (left panel), and PG and Δ2-PG (right panel) onPC-3 cell viability. PC-3 cells were exposed to individual test agentsat the indicated concentrations in serum-free RPMI 1640 medium in96-well plates for 24 h, and cell viability was assessed by MTT assay.Each data point represents the mean±S.D. (n=6). For the analysis ofcytochrome c release, PC-3 cells were treated with the test agent at theindicated concentration for 24 h in serum-free RMPI 1640 medium for 24h, and mitochondria-free lysates were prepared. Equivalent amounts ofprotein from individual lysates were electrophoresed, and probed byWestern blotting with anti-cytochrome c antibody.

FIG. 4 Effect of TG on the expression levels of Bcl-2 family members inPC-3 cells. PC-3 cells were exposed to 30 μM TG in serum-free RPMI 1640medium for the indicated times. Equivalent amounts of protein from celllysates were electrophoresed, and probed by Western blotting withindividual antibodies (left panel). The bar graph depicts the relativeexpression levels, normalized to actin levels, at the indicated timeafter drug treatment.

FIG. 5 Differential inhibition of B113 domain-mediated proteininteractions of Bak BH3 peptide with Bcl-xL or Bcl-2 by TZDs and theirΔ2-derivatives. (A) Displacement of Flu-BakBH3 peptide from Bcl-xL orBcl-2 by TG (left panel) and Δ2-TG (right panel). (B) IC₅₀ values ofindividual TZDs and Δ2-TZDs for inhibiting the BH3-mediated proteininteractions.

FIG. 6 TG and Δ2-TG trigger caspase-dependent apoptotic death byinhibiting heterodimer formation of Bcl-2 and Bcl-xL with Bak. (A)Effect of TG and Δ2-TO on the dynamics of Bcl-2/Bak (left panel) andBcl-xL/Bak (right panel) interactions in PC-3 cells. PC-3 cells weretreated; with 50 μM TG or Δ2-TG for 12 h, and cell lysates wereimmunoprecipitated with anti-Bcl-2 or anti-Bcl-xL antibodies. Theimmunoprecipitates were probed with anti-Bak antibodies by Western blotanalysis (WB) as described in the Materials and Methods. The bar graphsindicate the relative Bak levels, normalized to IgG light chains levels,in the three treatments. Values are means±S.D. (n=3). *P<0.01. (B)Dose-dependent effect of TG and Δ2-TG on caspase-9 activation in PC-3cells. PC-3 cells were treated with TG or Δ2-TG at the indicatedconcentrations for 24 h. Caspase-9 antibodies recognize the largesubunits (35 and 37 kDa). (C) Protection of TG and Δ2-TG-inducedapoptosis in PC-3 cells by the pan-caspase inhibitor Z-VAD-FMK. PC-3cells were pretreated with 100 μM Z-VAD-FMK 30 minute before exposure to30 μM TG or Δ2-TG in serum-free RPMI 1640 medium in 96-well plates for24 h, and cell viability was assessed by MTT assay. Each data pointrepresents the mean±S.D. (n=6). *P<0.01.

FIG. 7 Ectopic Bcl-xL protects LNCaP cells from TG- and Δ2-TG-inducedapoptosis by attenuating cytochrome c release in an expressionlevel-dependent manner. (A) Left panel, ascending expression levels ofectopic Bcl-xL in B11, B1, and B3 clones. Values are means±S.D. (n=3).*P<0.01. Right panel, Western blot analysis. The band for ectopic Bcl-xLcontained a FLAG tag (8 amino acids long) from the construct, thusmigrating slower than endogenous Bcl-xL. (B) Dose-dependent effects ofTG (left panel) and Δ2-TG (right panel) on apoptosis in LNCaP, B11, B1,and B3 cells. Data are mean±S.D. (n=3). (C) Ectopic expression of Bcl-xLinhibits the effect of TG (50 μM) and Δ2-TG (50 μM) on cytochrome crelease. Cells were treated with DMSO vehicle (−) or with 50 μM TG orΔ2-TG. Cytosol-specific, mitochondria-free lysates were prepared.Equivalent amounts of protein from individual lysates wereelectrophoresed, and probed by Western blotting with anti-cytochrome cantibody. The relative ratios of cytoplasmic cytochrome c indrug-treated to vehicle-treated cells are shown below the Western blots.Values are means±S.D. (n=3).

FIG. 8 Effect of oral TG-88 at 100 and 200 mg/kg on the growth of PC-3tumors in nude mice. Each mouse was inoculated subcutaneously in theright flank with 5×10⁵ PC-3 cells suspended in 0.1 ml of serum-freemedium containing 30% Matrigel under isoflurane anesthesia. Forty-eighthours later, mice were randomly divided into three groups (n=8) and wereadministered daily TG88 at 100 and 200 mg/kg body weight/day by gavagefor the duration of the study. Controls received vehicle consisting of0.5% methylcellulose and 0.1% polysorbate 80 in sterile water. Valuesare means±SE (n=8). *P<0.05 as compared to the control group.

DETAILED DESCRIPTION OF THE INVENTION

Provided are Bc1-xL/Bcl-2 binding inhibitors useful in the treatment ofunwanted proliferating cells, including cancers and precancers, insubjects in need of such treatment. Also provided are methods oftreating a subject having unwanted proliferating cells comprisingadministering a therapeutically effective amount of a compound describedherein to a subject in need of such treatment. Also provided are methodsof preventing the proliferation of unwanted proliferating cells, such ascancers and precancers, in a subject comprising the step ofadministering a therapeutically effective amount of a compound describedherein to a subject at risk of developing a condition characterized byunwanted proliferating cells. In some embodiments, the compoundsdescribed herein reduce the proliferation of unwanted cells by inducingapoptosis in those cells. In one embodiment, the compounds describedherein are used in the treatment of prostate cancer in a subject in needof such treatment. In another embodiment, the compounds described hereinare used in a method of preventing prostate cancer in a subject, whereinthe subject is at risk of developing prostate cancer. In someembodiments, the methods treating unwanted proliferating cells,including cancers and precancers, comprise inducing apoptosis in theunwanted proliferating cells by administering an effective amount of theBcl-xL/Bcl-2 binding inhibitors described herein to the subject in needof such treatment.

In one embodiment the Bcl-xL/Bcl-2 binding inhibitors described hereinhave the following structure:

wherein R is selected from aryl, heteroaryl, cycloalkyl,heterocycloalkyl, alkylaryl, and combinations thereof; and wherein R maybe substituted at one or more substitutable positions with a hydroxyl,or alkyl substituent. In some embodiments, R is selected from the groupconsisting of

Some embodiments include:

TABLE 1 Entry compound R IC50 for MTT IC50 for WB 1 Δ2-TG

57 22 2 Δ2-CG

70 13 3 Δ2-PG

4 TG-15

37 3.8

In another embodiment, the Bcl-xL/Bcl-2 binding inhibitors describedherein have the following structure:

wherein X₁ is selected from the group consisting of H, alkyl, alkoxy,halo, nitro, and combinations thereof; and X₂ is selected from the groupconsisting of H, alkyl, alkoxy, halo, and combinations thereof. In someembodiments, X₁ is selected from H, Br, CH₃, OCH₃, OCH₂CH₃, NO₂, and Cl;and X₂ is selected from H, CH₃, OCH₃, and Br. Some embodiments include:

TABLE 2 IC50 IC50 Entry compound X1 X2 for MTT for WB 5 TG-6 H H 9 3 6TG-27 Br H 28 >7.5 7 TG-28 OMe H 14.5 2.3 8 TG-29 Me H 23.5 3.6 9 TG-52Me Me 10.5 7.5 10 TG-54 Br OMe 17.5 3.8 11 TG-55 OEt H 17 >7.5 12 Br Br13 NO₂ H 14 Cl H

In another embodiment, the Bcl-xL/Bcl-2 binding inhibitors describedherein have the following structure:

wherein X₁ is selected from the group consisting of H, alkyl, alkoxy,halo, nitro, haloalkylaryl, haloaryl, alkylaryl, and combinationsthereof.; and X₂ is selected from the group consisting of H, alkyl,alkoxy, halo, and combinations thereof. In some embodiments, X₁ isselected from the group consisting of H, methyl, methoxy, ethoxy,fluoro, chloro, bromo, nitro, trifluoromethylphenyl, fluorophenyl, andethylphenyl; and X₂ is selected from the group consisting of H, methyl,methoxy, and bromo. Some embodiments are shown in the table below.

TABLE 3 IC50 IC50 Entry compound X1 X2 for MTT for WB 15 TG-14 H H 14.57 16 TG-16 OMe H 15 5.6 17 TG-17 Me H 14.5 3.2 18 TG-30 F H 12.5 7.2 19TG-31

H >50 >7.5 20 TG-32

H 19.5 >7.5 21 TG-33

H >50 >7.5 22 TG-34 Br Br 15.5 2.7 23 TG-35 N₂O H 38 2.7 24 TG-44 Br OMe14.5 >7.5 25 TG-45 OEt H 13 6.7 26 TG-88 Br H 14.5 >7.5 27 Me Me 28 Cl H

In another embodiment, the Bcl-xL/Bcl-2 binding inhibitors describedherein have the following structure:

wherein X₁ is selected from the group consisting of H, halo, andcombinations thereof and Y is selected from the group consisting ofalkylaryl, ankenylaryl, alkenyl, ester carboxylic acids, ester alcohols,and combinations thereof. In some embodiments, X₁ is selected from thegroup consisting of H and Br, and Y is selected from the groupconsisting of

Some embodiments are shown in the table below.

TABLE 4 com- IC50 IC50 Entry pound X1 Y for MTT for WB 29 TG-10 H

19 >7.5 30 TG-11 Br

28.5 >7.5 31 TG-12 H

16.67 3.6 32 TG-13 H

>50 >7.5 33 Br

34 H

35 Br

36 H

37 Br

38 Br

In another embodiment, the Bcl-xL/Bcl-2 binding inhibitors describedherein have the following structure:

wherein X₁ is selected from the group consisting of H, halo, andcombinations thereof; and Y is selected from the group consisting ofstraight-chain alkenyl, branched alkenyl, and combinations thereof. Somespecific embodiments include:

TABLE 5 IC50 Entry compound X1 Y IC50 for MTT for WB 39 TG-3 H

11 3.5 40 TG-89 Br

In another embodiment, the Bcl-xL/Bcl-2 binding inhibitors describedherein have the following structure:

wherein X₁ is selected from the group consisting of H, alkoxy, halo, andcombinations thereof; and Z is selected from the group consisting of

and combinations thereof. Some specific embodiments include:

TABLE 6 Entry compound X1 Z IC50 for MTT IC50 for WB 41 TG-9 H

>50 >7.5 42 H

43 OMe

44 OEt

45 H

46 OMe

47 OEt

48 H

49 OMe

50 OEt

51 H

52 OMe

53 OEt

54 H

55 OMe

56 OEt

57 H

58 OMe

59 OEt

In another embodiment, the Bcl-xL/Bcl-2 binding inhibitors describedherein have the following structure:

wherein X₁ is selected from the group consisting of H, halo, andcombinations thereof and Z is selected from the group consisting of

and combinations thereof. Some specific embodiments are shown in thetable, below:

TABLE 7 Entry compound X1 Z IC50 for MTT IC50 for WB 60 TG-36 Br

>50 >7.5 61 TG-37 Br

34 >7.5 62 TG-38 Br

>50 4.5 63 TG-39 Br

46 >7.5 64 TG-41 Br

>50 >7.5 65 TG-42 Br

>50 >7.5 66 H

67 Br

68 Cl

In another embodiment, the Bcl-xL/Bcl-2 binding inhibitors describedherein have the following structure:

wherein W is selected from O, S and combinations thereof; Y is selectedfrom straight chain alkenyl, branched alkenyl and combinations thereof,and Z′ is selected from H and carboxylic acid. Some specific embodimentsare shown in the table below:

TABLE 8 Entry compound W Y Z′ IC50 for MTT IC50 for WB 69 TG-43 O

H 14.5 7.2 70 TG-46 S

H 37.33 6.7 71 TG-53 O

H 14.5 3.4 72 O

In another embodiment, the Bcl-xL/Bcl-2 binding inhibitors describedherein have the following structure:

wherein Y is selected from straight chain alkenyl, branched alkenyl andcombinations thereof. Some specific embodiments are shown in the tablebelow:

TABLE 9 Entry compound Y IC50 for MTT IC50 for WB 73 TG-51

40 4.4 74

In another embodiment, the Bcl-xL/Bcl-2 binding inhibitors describedherein have the following structure:

wherein Y is selected from straight chain alkenyl, branched alkenyl andcombinations thereof, and Z′ is selected from H and carboxylic acid.Some specific embodiments are shown in the table below:

TABLE 10 Entry Y Z′ 75

H 76

77

H

In another embodiment, the Bcl-xL/Bcl-2 binding inhibitors describedherein have the following structure:

wherein Y is selected from straight chain alkenyl, branched alkenyl andcombinations thereof, and Z′ is selected from H and carboxylic acid.Some specific embodiments are shown in the table below:

TABLE 11 Entry Y Z′ 78

H 79

80

H

Abbreviations used herein: PPARγ, peroxisome proliferator-activatedreceptor γ; TZDs, thiazolidenediones; TG, troglitazone; CG, ciglitazone;RG, rosiglitazone; PG, pioglitazone; Δ2-TG,5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-benzylidene]-2,4-thiazolidinedione;5-[4-(1-methyl-cyclohexylmethoxy)-benzylidene]-thiazolidine-2,4-dione,Δ2-RG,5-{4-[2-(methyl-pyridin-2-yl-amino)-ethoxy]-benzylidene}-thiazolidine-2,4-dione,5-{4-[2-(5-ethyl-pyridin-2-yl)-ethoxy]-benzylidene}-thiazolidine-2,4-dione;FBS, fetal bovine serum; MTT,[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide]; FP,fluorescence polarization.

As used herein, the term “prevention” includes either preventing theonset of a clinically evident unwanted cell proliferation altogether orpreventing the onset of a preclinically evident stage of unwanted rapidcell proliferation in individuals at risk. Also intended to beencompassed by this definition is the prevention of metastasis ofmalignant cells or to arrest or reverse the progression of malignantcells. This includes prophylactic treatment of those at risk ofdeveloping precancers and cancers.

The terms “therapeutically effective” and “pharmacologically effective”are intended to qualify the amount of each agent which will achieve thegoal of improvement in disease severity and the frequency of incidenceover treatment of each agent by itself, while avoiding adverse sideeffects typically associated with alternative therapies.

The term “subject” for purposes of treatment includes any human oranimal subject who has a disorder characterized by unwanted, rapid cellproliferation. Such disorders include, but are not limited to cancersand precancers. For methods of prevention the subject is any human oranimal subject, and preferably is a human subject who is at risk ofobtaining a disorder characterized by unwanted, rapid cellproliferation, such as cancer. The subject may be at risk due toexposure to carcinogenic agents, being genetically predisposed todisorders characterized by unwanted, rapid cell proliferation, and soon. Besides being useful for human treatment, the compounds of thepresent invention are also useful for veterinary treatment of mammals,including companion animals and farm animals, such as, but not limitedto dogs, cats, horses, cows, sheep, and pigs. In most embodiments,subject means a human.

The phrase “pharmaceutically acceptable salts” connotes salts commonlyused to form alkali metal salts and to form addition salts of free acidsor free bases. The nature of the salt is not critical, provided that itis pharmaceutically acceptable. Suitable pharmaceutically acceptableacid addition salts of compounds of formulae I and II may be preparedfrom an inorganic acid or from an organic acid. Examples of suchinorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric,carbonic, sulfuric, and phosphoric acid. Appropriate organic acids maybe selected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic, and sulfonic classes of organic acids,examples of which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucoronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic,salicylic, p-hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic,methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, galactaric,and galacturonic acids. Suitable pharmaceutically acceptable baseaddition salts of the compounds described herein include metallic saltsmade from aluminum, calcium, lithium, magnesium, potassium, sodium, andzinc. Alternatively, organic salts made fromN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine may be usedform base addition salts of the compounds described herein. All of thesesalts may be prepared by conventional means from the correspondingcompounds described herein by reacting, for example, the appropriateacid or base with the compound.

Where the term alkyl is used, either alone or with other terms, such ashaloalkyl or alkylaryl, it includes C₁ to C₁₀ linear or branched alkylradicals, examples include methyl, ethyl, propyl, isopropyl, butyl,tert-butyl, and so forth. The term “haloalkyl” includes C₁ to C₁₀ linearor branched alkyl radicals substituted with one or more halo radicals.Some examples of haloalkyl radicals include trifluoromethyl,1,2-dichloroethyl, 3-bromopropyl, and so forth. The term “halo” includesradicals selected from F, Cl, Br, and I. Alkyl radical substituents ofthe present invention may also be substituted with other groups such asazido, for example, azidomethyl, 2-azidoethyl, 3-azidopropyl and so on.

The term aryl, used alone or in combination with other terms such asalkylaryl, haloaryl, or haloalkylaryl, includes such aromatic radicalsas phenyl, biphenyl, and benzyl, as well as fused aryl radicals such asnaphthyl, anthryl, phenanthrenyl, fluorenyl, and indenyl and so forth.The term “aryl” also encompasses “heteroaryls,” which are aryls thathave carbon and one or more heteroatoms, such as O, N, or S in thearomatic ring. Examples of heteroaryls include indolyl, pyrrolyl, and soon. “Alkylaryl” or “arylalkyl” refers to alkyl-substituted aryl groupssuch as butylphenyl, propylphenyl, ethylphenyl, methylphenyl,3,5-dimethylphenyl, tert-butylphenyl and so forth. “Haloaryl” refers toaryl radicals in which one or more substitutable positions has beensubstituted with a halo radical, examples include fluorophenyl,4-chlorophenyl, 2,5-chlorophenyl and so forth. “Haloalkylaryl” refers toaryl radicals that have a haloalkyl substituent.

Provided are pharmaceutical compositions for ablating cyclin D1 in MCF-7cells specifically. These compounds are also useful for treating,preventing, or delaying the onset of a cancer in a subject in need ofsuch treatment. The pharmaceutical composition comprises atherapeutically effective amount of a compound disclosed herein, or aderivative or pharmaceutically acceptable salt thereof, in associationwith at least one pharmaceutically acceptable carrier, adjuvant, ordiluent (collectively referred to herein as “carrier materials”) and, ifdesired, other active ingredients. The active compounds of the presentinvention may be administered by any suitable route known to thoseskilled in the art, preferably in the form of a pharmaceuticalcomposition adapted to such a route, and in a dose effective for thetreatment intended. The active compounds and composition may, forexample, be administered orally, intra-vascularly, intraperitoneally,intranasal, intrabronchial, subcutaneously, intramuscularly or topically(including aerosol). With some subjects local administration, ratherthan system administration, may be preferred. Formulation in a lipidvehicle may be used to enhance bioavailability.

The administration of the present invention may be for either preventionor treatment purposes. The methods and compositions used herein may beused alone or in conjunction with additional therapies known to thoseskilled in the art in the prevention or treatment of disorderscharacterized by unwanted, rapid proliferation of cells. Alternatively,the methods and compositions described herein may be used as adjuncttherapy. By way of example, the compounds of the present invention maybe administered alone or in conjunction with other antineoplastic agentsor other growth inhibiting agents or other drugs or nutrients, as in anadjunct therapy.

The phrase “adjunct therapy” or “combination therapy” in defining use ofa compound described herein and one or more other pharmaceutical agents,is intended to embrace administration of each agent in a sequentialmanner in a regimen that will provide beneficial effects of the drugcombination, and is intended as well to embrace co-administration ofthese agents in a substantially simultaneous manner, such as in a singleformulation having a fixed ratio of these active agents, or in multiple,separate formulations for each agent.

For the purposes of combination therapy, there are large numbers ofantineoplastic agents available in commercial use, in clinicalevaluation and in pre-clinical development, which could be selected fortreatment of cancers or other disorders characterized by rapidproliferation of cells by combination drug chemotherapy. Suchantineoplastic agents fall into several major categories, namely,antibiotic-type agents, alkylating agents, antimetabolite agents,hormonal agents, immunological agents, interferon-type agents and acategory of miscellaneous agents. Alternatively, other anti-neoplasticagents, such as metallomatrix proteases inhibitors (MMP), such as MMP-13inhibitors, or β_(ν)β₃ inhibitors may be used. Suitable agents which maybe used in combination therapy will be recognized by those of skill inthe art. Similarly, when combination therapy is desired, radioprotectiveagents known to those of skill in the art may also be used.

When preparing the compounds described herein for oral administration,the pharmaceutical composition may be in the faun of, for example, atablet, capsule, suspension or liquid. The pharmaceutical composition ispreferably made in the faun of a dosage unit containing a particularamount of the active ingredient. Examples of such dosage units arecapsules, tablets, powders, granules or a suspension, with conventionaladditives such as lactose, mannitol, corn starch or potato starch; withbinders such as crystalline cellulose, cellulose derivatives, acacia,corn starch or gelatins; with disintegrators such as corn starch, potatostarch or sodium carboxymethyl-cellulose; and with lubricants such astalc or magnesium stearate. The active ingredient may also beadministered by injection as a composition wherein, for example, saline,dextrose or water may be used as a suitable carrier.

For intravenous, intramuscular, subcutaneous, or intraperitonealadministration, the compound may be combined with a sterile aqueoussolution which is preferably isotonic with the blood of the recipient.Such formulations may be prepared by dissolving solid active ingredientin water containing physiologically compatible substances such as sodiumchloride, glycine, and the like, and having a buffered pH compatiblewith physiological conditions to produce an aqueous solution, andrendering said solution sterile. The formulations may be present in unitor multi-dose containers such as sealed ampoules or vials.

For treating cancers or other unwanted proliferative cells that arelocalized in the G.I. tract, the compound may be formulated withacid-stable, base-labile coatings known in the art which begin todissolve in the high pH small intestine. Formulation to enhance localpharmacologic effects and reduce systemic uptake are preferred.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active compound which ispreferably made isotonic. Preparations for injections may also beformulated by suspending or emulsifying the compounds in non-aqueoussolvent, such as vegetable oil, synthetic aliphatic acid glycerides,esters of higher aliphatic acids or propylene glycol.

The dosage form and amount can be readily established by reference toknown treatment or prophylactic regiments. The amount of therapeuticallyactive compound that is administered and the dosage regimen for treatinga disease condition with the compounds and/or compositions of thisinvention depends on a variety of factors, including the age, weight,sex, and medical condition of the subject, the severity of the disease,the route and frequency of administration, and the particular compoundemployed, the location of the unwanted proliferating cells, as well asthe pharmacokinetic properties of the individual treated, and thus mayvary widely. The dosage will generally be lower if the compounds areadministered locally rather than systemically, and for prevention ratherthan for treatment. Such treatments may be administered as often asnecessary and for the period of time judged necessary by the treatingphysician. One of skill in the art will appreciate that the dosageregime or therapeutically effective amount of the inhibitor to beadministrated may need to be optimized for each individual. Thepharmaceutical compositions may contain active ingredient in the rangeof about 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mgand most preferably between about 1 and 200 mg. A daily dose of about0.01 to 100 mg/kg body weight, preferably between about 0.1 and about 50mg/kg body weight, may be appropriate. The daily dose can beadministered in one to four doses per day.

Several embodiments of the Bcl-xL/Bcl-2 inhibitors described herein,along with IC₅₀ values for Bcl-xL/Bak, Bcl-2/Bak and PC3 cells are shownin Table 12, below.

TABLE 12 IC₅₀ IC₅₀ IC₅₀ (μM) Entry cmpd. Bcl-xl/Bak Bcl-2/Bak PC3structure 1 Δ2-TG 18 ± 1  18 ± 1  20 ± 2 

2 Δ2-CG 17 ± 2  22 ± 3   15 ± 1.2

3 Δ2-PG >50 >50 >50

4 TG-6 6.0 ± 0.5 5.5 ± 0.4  11 ± 0.8

5 TG-3 4.8 ± 0.5 7.3 ± 0.3  10 ± 1.2

6 TG-9 >50 >50 >50

7 TG-10  13 ± 0.9 19 ± 0.8  19 ± 0.5

8 TG-11 2.2 ± 0.2 3.1 ± 0.4  6 ± 0.3

9 TG-12  14 ± 1.1  18 ± 0.9  18 ± 1.3

10 TG-13  28 ± 1.4  30 ± 1.5  22 ± 0.6

11 TG-14 4.5 ± 0.4 4.8 ± 0.4   9 ± 1.0

12 TG-15  16 ± 0.5  18 ± 0.9  12 ± 0.9

13 TG-16 6.5 ± 0.5 4.8 ± 0.5 4.7 ± 0.2

14 TG-17 2.6 ± 0.3 3.1 ± 0.4 4.8 ± 0.3

15 TG-27 2.1 ± 0.2 2.7 ± 0.4 5.0 ± 0.5

16 TG-28 4.0 ± 0.3 4.4 ± 0.3 5.2 ± 0.5

17 TG-29 3.0 ± 0.3 3.5 ± 0.6 5.1 ± 0.4

18 TG-30 2.7 ± 0.3 3.4 ± 0.5 6.0 ± 0.4

19 TG-31 4.9 ± 1.2 4.7 ± 2.0  30 ± 1.1

20 TG-32 6.5 ± 0.5 6.6 ± 0.6 5.0 ± 0.4

21 TG-33  18 ± 0.8  21 ± 1.4  17 ± 0.9

22 TG-34 2.0 ± 0.2 2.8 ± 0.4 4.4 ± 0.4

23 TG-35 >50

24 TG-36 >50

25 TG-37   6 ± 0.7

26 TG-38 12.5 ± 0.6 

27 TG-39   7 ± 0.4

28 TG-41 >50

29 TG-42  16 ± 0.3

30 TG-43 3.0 ± 0.2

31 TG-44 5.1 ± 0.4

32 TG-45 4.6 ± 0.3

33 TG-46 5.3 ± 0.3

34 TG-51 6.8 ± 0.2

35 TG-52 4.5 ± 0.2

36 TG-53 3.3 ± 0.3

37 TG-54

38 TG-55

39 TG-88 1.8 ± 0.2 2.8 ± 0.3 4.2 ± 0.2

40 TG-89 4.4 ± 0.4

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

Materials and Methods

Reagents. Troglitazone (TG) and ciglitazone (CG) were purchased fromSigma (St. Louis, Mo.) and Cayman Chemical (Ann Arbor, Mich.),respectively. Rosiglitazone (RG) and pioglitazone (PG) were preparedfrom the respective commercial capsules by solvent extraction followedby recrystallization or chromatographic purification. Δ2-TG{5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-benzylidene]-2,4-thiazolidine-dione},Δ2-CG{5-[4-(1-methyl-cyclohexylmethoxy)-benzylidene]-thiazolidine-2,4-dione},Δ2-RG{5-{4-[2-(methyl-pyridin-2-yl-amino)-ethoxy]-benzylidene}-thiazolidine-2,4-dione},Δ2-PG{5-{4-[2-(5-ethyl-pyridin-2-yl)-ethoxy]-benzylidene}-thiazolidine-2,4-dione}(FIG. 1A), and TG-88 are TZD derivatives with attenuated orunappreciable activity in PPARγ activation, of which the synthesis willbe published elsewhere. The identity and purity (≧99%) of thesesynthetic derivatives were verified by proton nuclear magneticresonance, high-resolution mass spectrometry, and elemental analysis.For in vitro experiments, these agents at various concentrations weredissolved in DMSO, and were added to cells in medium with a final DMSOconcentration of 0.1%. For the in vivo study, TG88 was prepared as asuspension by sonication in a vehicle consisting of 0.5% methylcelluloseand polysorbate 80 in sterile water. The pan-caspase inhibitor Z-VAD-FMKwas purchased from BD Bioscience. The Cell Death Detection ELISA kit waspurchased from Roche Diagnostics (Mannheim, Germany). The NuclearExtract kit and PPARγ Transcription Factor Assay kit were obtained fromActive Motif (Carlsbad, Calif.). Rabbit antibodies against Bcl-xL, Bax,Bak, Bid, and cleaved caspase-9 were purchased from Cell SignalingTechnology Inc. (Beverly, Mass.). Rabbit antibodies against Bad, andcytochrome c, and mouse anti-Bcl-2, anti-.-tubulin were from Santa CruzBiotechnology, Inc. (Santa Cruz, Calif.). Mouse monoclonal anti-actinwas from ICN Biomedicals Inc (Costa Mesa, Calif.). Goat anti-rabbitimmunoglobulin G (IgG)-horseradish peroxidase conjugates and rabbitanti-mouse IgG horseradish peroxidase conjugates were from JacksonImmunoResearch Laboratories (West Grove, Pa.). 6C8 Hamster anti-humanBcl-2 antibody for immunoprecipitation was purchased from Pharmingen(San Diego, Calif.).

Cell Culture. LNCaP androgen-dependent (p53^(+/+)) and PC-3androgen-nonresponsive (P53^(−/−)) prostate cancer cells were obtainedfrom the American Type Culture Collection (Manassas, Va.), and weremaintained in RPMI 1640 medium supplemented with 10% fetal bovine serum(FBS) at 37° C. in a humidified incubator containing 5% CO₂. Preparationof the stable Bcl-xL-overexpressing LNCaP clones B11, B1, and B3 werepreviously described (16).

Cell viability analysis. The effect of individual test agents on cellviability was assessed by using the MTT{[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide]}assay in six to twelve replicates. Cells were seeded and incubated in96-well, flat-bottomed plates in serum-free media for 24 h, and wereexposed to various concentrations of test agents dissolved in DMSO(final concentration, 0.1%) in serum-free RPMI 1640 medium for differenttime intervals. Controls received DMSO vehicle at a concentration equalto that in drug-treated cells. The medium was removed, replaced by 200μl of 0.5 mg/ml of MTT in 10% FBS-containing RPMI-1640 medium, and cellswere incubated in the CO₂ incubator at 37° C. for 2 h. Supernatants wereremoved from the wells, and the reduced MTT dye was solubilized in 200DMSO. Absorbance at 570 nm was determined on a plate reader.

Apoptosis Detection by An Enzyme-Linked Immunosorbent Assay (ELISA).Induction of apoptosis was assessed with a Cell Death Detection ELISAkit (Roche Diagnostics, Mannheim, Germany) by following themanufacturer's instruction. This test is based on the quantitativedetermination of cytoplasmic histone-associated DNA fragments in theform of mononucleosomes and oligonucleosomes after induced apoptoticdeath. In brief, 1×10⁶ cells were cultured in a T-25 flask in 10%FBS-containing medium for 24 h, and were treated with the test agents atvarious concentrations in serum-free medium for 24 h. Both floating andadherent cells were collected, cell lysates equivalent to 5×10⁵ cellswere used in the ELISA.

Western Blot Analysis of Cytochrome c Release into the Cytoplasm.Cytosolic-specific, mitochondria-free lysates were prepared according toan established procedure (16). In brief, after individual treatments for24 h, both the incubation medium and adherent cells in T-75 flasks werecollected, and centrifuged at 200×g for 5 min. The pellet fraction wasrecovered, placed on ice, and triturated with 300 μl of a chilledhypotonic lysis solution [50 mM PIPES-KOH, pH 7.4, containing 220 mMmannitol, 68 mM sucrose, 50 mM KCl, 5 mM EDTA, 2 mM MgCl₂, 1 mM DTT, anda mixture of protease inhibitors including 100 μM AEBSF, 80 nMaprotinin, 5 μM bestatin, 1.5 μM E-64 protease inhibitor, 2 μMleupeptin, and 1 μM pepstatin A]. After a 45 min-incubation on ice, themixture was centrifuged at 200×g for 10 min. The supernatant wascollected in a microcentrifuge tube, and centrifuged at 14,000 rpm for30 min. An equivalent amount of protein (50 μg) from each supernatantwas resolved in 10% SDS-polyacrylamide gel. Bands were transferred tonitrocellulose membranes, and analyzed by immunoblotting withanti-cytochrome c antibodies, as described below.

Immunoblotting. Cells in T-75 flasks were collected by scraping, andsuspended in 60 μl of phosphate-buffered saline (PBS). Two μl of thesuspension was taken for protein analysis using the Bradford assay kit(Bio-Rad, Hercules, Calif.). To the remaining solution was added thesame volume of 2×SDS-PAGE sample loading buffer (100 mM Tris-HCl, pH6.8, 4% SDS, 5% β-mercaptoethanol 20% glycerol, and 0.1% bromophenolblue). The mixture was sonicated briefly, and boiled for 5 min. Equalamounts of proteins were loaded onto 10% SDS-PAGE gels.

After electrophoresis, protein bands were transferred to nitrocellulosemembranes in a semidry transfer cell. The transblotted membrane waswashed twice with Tris-buffered saline (TBS) containing 0.1% Tween 20(TBST). After blocking with TBST containing 5% nonfat milk for 40 min,the membrane was incubated with the appropriate primary antibody inTBST-1% nonfat milk at 4° C. overnight. All primary antibodies werediluted 1:1000 in 1% nonfat milk-containing TBST. After treatment withthe primary antibody, the membrane was washed three times with TBST fora total of 15 min, followed by incubation with goat anti-rabbit oranti-mouse IgG-horseradish peroxidase conjugates (diluted 1:5000) for 1h at room temperature and three washes with TBST for a total of 1 h. Theimmunoblots were visualized by enhanced chemiluminescence.

Analysis of PPARγ activation. The analysis was carried out by using aPPARγ transcription factor ELISA kit (Active Motif, Carlsbad, Calif.),in which an oligonucleotide containing the peroxisome proliferatorresponse element (PPRE) was immobilized onto a 96-well plate. PPARscontained in nuclear extracts bind specifically to this oligonucleotideand are detected through an antibody directed against PPARγ. In brief,PC-3 cells were cultured in RPMI 1640 medium supplemented with 10% FBS,and treated with DMSO vehicle or individual test agents, 10 μM each, for48 h. Cells were collected, and nuclear extracts were prepared with aNuclear Extract kit (Active Motif, Carlsbad, Calif.). Nuclear extractsof the same protein concentration from individual treatments weresubject to the PPARγ transcription factor ELISA according to themanufacturer's instruction.

Competitive Fluorescence Polarization Assay. The binding affinity of thetest agent to Bcl-2 and Bcl-xL was analyzed by a competitivefluorescence polarization assay, in which the ability of the agent todisplace the binding of a Bak BH3-domain peptide to either Bcl-2 orBcl-xL was determined. Flu-BakBH3, a Bak-BH3 peptide labeled at theN-terminus with fluorescein, was purchased from Genemed Synthesis (SanFrancisco, Calif.). C-Terminal-truncated, His-tagged Bcl-x_(L) waspurchased from EMD Biosciences (San Diego, Calif.), and solubleGST-fused Bcl-2 was obtained from Santa Cruz (Santa Cruz, Calif.). TheK_(D) determination was carried out in a dual-pathlength quartz cellwith readings taken at λ_(em) 480 nm and λ_(ex) 530 nm at roomtemperature using a luminescence spectrometer according to anestablished procedure (17).

Determination of IC₅₀ values. Data from cell viability and FP assayswere analyzed by using the CalcuSyn software (Biosoft, Ferguson, Mo.) todetermine IC₅₀ values, in which the calculation was based on the mediumeffect equation (18), i.e., Log(fa/fu)=m log(D)−m log(Dm) (equation 1),where fa and fu denote fraction affected and unaffected, respectively; mrepresents the Hill-type coefficient signifying the sigmoidicity of thedose-effect curve; D and Dm are the dose used and IC₅₀, respectively.

Co-immunoprecipitation. PC3 cells treated with 50 μM TG or Δ2-TG for 12hr were collected, and lysed by NP-40 isotonic lysis buffer with freshlyadded protease inhibitors (142 mM KCl, 5 mM MgCl₂, 10 mM HEPES, pH 7.2,1 mM EGTA, 0.2% NP-40, 0.2 mM PMSF, and 1 μg/ml, each aprotinin,leupeptin, and pepstatin). After centrifugation at 13000×g for 15 min,the supernatants were collected, pre-incubated with protein A-Sepharose(Sigma, St. Louis, Mo.) for 15 min, and centrifuged at 1000×g for 5 min.The supernatants were exposed to Bcl-2 or Bcl-xL antibodies in thepresence of protein A-Sepharose at 4° C. for 2 h. After briefcentrifugation, protein A-Sepharose were collected, washed with theaforementioned lysis buffer 2 times, suspended in 2×SDS sample buffer,and subjected to Western blot analysis with antibodies against Bak.

Xenograft tumor growth. Male NCr athymic nude mice (5-7 weeks of age)were obtained from the National Cancer Institute (Frederick, Md.). Themice were group-housed under conditions of a constant 12-h photoperiodwith ad libitum access to sterilized food and water. All experimentalprocedures utilizing these mice were performed in accordance withprotocols approved by the Institutional Laboratory Animal Care and UseCommittee of The Ohio State University.

Each mouse was inoculated subcutaneously in the right flank with 5×10⁵PC-3 cells suspended in 0.1 ml of serum-free medium containing 30%Matrigel (BD Biosciences, Bedford, Mass.) under isoflurane anesthesia.Forty-eight hours later, mice were randomly divided into three groups(n=8) and were administered daily TG88 at 100 and 200 mg/kg bodyweight/day by gavage for the duration of the study. Controls receivedvehicle consisting of 0.5% methylcellulose and 0.1% polysorbate 80 insterile water. The volume of drug or vehicle administered to each mousewas 0.02 ml/gram body weight. Tumors were measured weekly using calipersand their volumes calculated using a standard formula:width²×length×0.52. Body weights were measured weekly.

Statistical analysis. For in vitro studies, data were analyzed byone-way ANOVA followed Fisher's LSD for multiple comparisons. These dataare expressed as means±SD. For the in vivo study, tumor volume datafailed to meet the assumption of noituality (Shapiro-Wilk test) forparametric analysis; thus, group means were compared using theKruskall-Wallis ANOVA procedure and the Mann-Whitney U test. Tumorgrowth data are expressed as mean tumor volumes±SE. For all data,differences were considered significant at P<0.05. Statisticalprocedures were performed using SPSS for Windows (SPSS, Inc., Chicago,Ill.).

Results

Development of TZD derivatives lacking PPARγ ligand activity. It wasreported that introduction of a double bond adjoining the terminalthiazolidine-2,4-dione ring of RG abrogated its PPARγ ligand property(19). As part of our effort to discern the role of PPARγ activation inthe antitumor effects of TZDs, we synthesized this RG derivative and thecounterparts of TG, PG, and CG, and examined the ability of theresulting molecules (Δ2-TG, Δ2-RG, Δ2-PG, and Δ2-CG) vis-à-vis theirparent TZDs to activate PPARγ in PC-3 cells (FIG. 1).

Among these new compounds, Δ2-RG showed a 77% reduction in the activityin PPARγ activation as compared to RG, which is in line with thatreported in the literature (19). In contrast, Δ2-TG, Δ2-PG, and Δ2-CGwere completely devoid of the ligand binding activity since therespective levels of PPARγ activation were not statistically differentfrom that of the DMSO vehicle (P<0.01). The loss/attenuation of PPARγactivity in these Δ2-derivatives was presumably attributable to thestructural rigidity, as a result of the double bond introduction,surrounding the heterocyclic system.

Apoptosis-inducing effects of TZDs on prostate cancer cells areindependent of PPARγ activation. We first assessed the dose-dependentgrowth inhibitory effect of TG and Δ2-TG in two prostate cancer celllines, androgen-independent PC-3 (p53^(−/−)) and androgen-dependentLNCaP (p53^(+/+)). Among many genotypic differences, these two celllines exhibit distinct PPARγ expression status (15), i.e., PPARγ washighly expressed in PC-3 cells, but was deficient in LNCaP cells (FIG.2A; P<0.01). Nevertheless, despite deficiency in PPARγ, LNCaP cellsexhibited a higher degree of susceptibility to TG-mediated in vitroantitumor effects as compared to the PPARγ-rich PC-3 cells (FIG. 2B). Inaddition, Δ2-TG, though devoid of PPARγ-activating activity, was morepotent than TG in suppressing cell proliferation in both cell lines. Therespective IC₅₀ values for TG and Δ2-TG were 30±2 and 20±2 μM in PC-3cells, and 22±3 and 14±1 μM in LNCaP cells. This growth inhibition wasattributable to apoptotic cell death, as evidenced by mitochondrialcytochrome c release and DNA fragmentation in PC-3 cells (FIG. 2C).Similar results were obtained with CG and Δ2-CG with respect tocytochrome c-dependent apoptotic death in PC-3 cells. The relativepotency paralleled that of TG and Δ2-TG (FIG. 3A). In contrast, RG, PG,and their Δ2-counterparts showed marginal effects, even at 50 μM, onapoptotic death in PC-3 cells (FIG. 3B). Together, these data suggestthat TZDs mediated apoptosis induction in prostate cancer cell systemsirrespective of PPARγ activation.

Apoptosis-active TZDs are inhibitors of Bcl-xL and Bcl-2 functions. Ourmechanistic study indicated that TG and Δ2-TG were able to sensitizePC-3 cells to the apoptosis-inducing effect of the phosphoinositide3-kinase (PI3K) inhibitor LY294002 (unpublished data). This finding,together with our recent report that attributed the resistance of PC-3cells to LY294002-induced apoptosis to Bcl-xL overexpression (16),suggests a plausible link between TZD-induced apoptosis and modulationof the functions of Bcl-xL and/or other Bcl-2 members.

Accordingly, we examined this putative link by two distinct approachesat both transcriptional and post-translational levels. First, weassessed the time-dependent effect of TG (30 μM) on the expression ofdifferent Bcl-2 family members in PC-3 cells, including Bcl-xL, Bcl-2,Bax, Bak, Bad, and Bid. This analysis was based on recent reports thattreatment of MCF-7 breast cancer and HepG2 hepatoma cells with highdoses of TG altered the expression levels of certain Bcl-2 members (9,14). Second, in light of the recent discovery of small-molecule Bcl-2 orBcl-xL inhibitors that disrupt BH3 domain-mediated interactions withproapoptotic Bcl-2 members (20-26), we investigated the in vitro effectsof TZDs and their Δ2-counterparts on the antiapoptotic function ofBcl-xL and Bcl-2. It is well understood that the ability of Bcl-xL andBcl-2 to form heterodimers with proapoptotic Bcl-2 members viaBH3-domain binding plays a key role in their antiapoptotic functions.Therefore, a well-established competitive fluorescence polarization (FP)analysis was used to examine the effects of TZDs on the binding of a BakBH3-domain peptide to Bcl-xL and Bcl-2.

FIG. 4 indicates that with the exception of a slight decrease in Badexpression at 24 h, the exposure of PC-3 cells to 30 μM TG did not causeappreciable change in the expression level of any of these Bcl-2 membersthroughout the course of investigation.

Nevertheless, data from the competitive FP analysis suggest that TG, CG,and their Δ2-counterparts inhibited the antiapoptotic functions ofBcl-xL and Bcl-2 by disrupting the BH3 domain-mediated interactions withproapoptotic Bcl-2 members. FIG. 5A depicts the ability of TG and Δ2-TGto displace the binding of a fluorescein-labeled Bak BH3 domain peptideto Bcl-xL and Bcl-2. It is noteworthy that both compounds inhibited theBH3 peptide binding to Bcl-xL and Bcl-2 with equal potency, a distinctdifference from many reported small-molecule inhibitors that showeddiscriminative affinity between these two antiapoptotic Bcl-2 members.The IC₅₀ values for the inhibition of Bak BH3 peptide binding to eitherBcl-xL or Bcl-2 were 22±1 and 18±1 μM, for TG and Δ2-TG, respectively(FIG. 5A). CG and Δ2-CG showed similar effects on the protein-proteininteractions with comparable IC₅₀ values (FIG. 5B). On the other hand,RG, z2-RG, PG, and Δ2-PG, which were ineffective in inducing apoptoticdeath even at high doses, showed poor inhibitory activities with IC₅₀significantly greater than 50

In light of the integral role of Bcl-2 members in the modulation ofmitochondrial integrity, these in vitro binding data suggest thatinterference of the ability of Bcl-2 and Bcl-xL to bind with theirproapoptotic Bcl-2 partners represented a major pathway for TG, Δ2-TG,and CG counterparts to exert their apoptotic action. To corroborate thispremise, we obtained two lines of evidence, 1) TG and Δ2-TG attenuatedthe binding of intracellular Bcl-2 and Bcl-xL to proapoptotic Bcl-2members, and 2) overexpression of Bcl-xL provided protection against thedrug-induced apoptosis.

Effect of TG and Δ2-TG on intracellular Bcl-2 and Bcl-xL binding to Bak.The functional relationship among different types of Bcl-2 familymembers in regulating the apoptosis machinery has been the focus of manyrecent investigations (27). One school of thought is that Bcl-2 andBcl-xL sequester Bax, Bak and other proapoptotic Bcl-2 members throughBH3 domain-mediated heterodimerization, thereby abrogating theirproapoptotic effects (28-32). For example, electrophoretic introductionof Bak or Bax BH3-domain peptides into PC-3 cells disrupted Bcl-2-Bakheterodimer formation, which resulted in the liberation of Bax and Bakto mediate apoptotic death via a caspase-dependent pathway (32).Consequently, to validate the mode of action of TG and Δ2-TG, weassessed the effects on the dynamics of Bcl-2/Bak and Bcl-xL/Bakinteractions in PC-3 cells. Lysates from PC-3 cells treated with TG orΔ2-TG (50 μM) vis-à-vis DMSO for 12 h were immunoprecipitated withantibodies against Bcl-2 or Bcl-xL. Probing of the immunoprecipitateswith anti-Bak antibodies by Western blotting indicates that the level ofBak associated with Bcl-2 and Bcl-xL was significantly reduced ascompared to the DMSO control (FIG. 6A; P<0.01). This decrease inintracellular associations bore out the in vitro binding data that TGand Δ2-TG inhibited the interactions of Bcl-xL and Bcl-2 with the BakBH3-domain peptide. We further demonstrated that treatment of PC-3 cellswith TG or Δ2-TG led to caspase-9 activation in a dose-dependent manner(FIG. 6B) similar to that of cytochrome c release (FIG. 2A).Furthermore, pretreatment of PC-3 cells with the pan-caspase inhibitorZ-VAD-FMK protected cells from TG- and Δ2-TG-induced apoptosis (FIG. 6C;P<0.01), confirming the involvement of caspase activation in apoptoticdeath.

Bcl-xL overexpression protects prostate cancer cells from TG- andΔ2-TG-Induced Apoptosis. We previously reported that LNCaP cellsexhibited lower Bcl-xL expression levels as compared to PC-3 cells (16),which underscored differences between these two cell lines in thesusceptibility to the apoptotic effects of TG, CG, and theirΔ2-counterparts. To confirm that the inhibition of Bcl-xL functionsplays a key role in the apoptosis induction, we examined the impact ofBcl-xL overexpression on the susceptibility to TG- and Δ2-TG-inducedcell death in LNCaP cells. Three transfected clones (B11, B1, and B3)that displayed ascending expression levels of Bcl-xL were testedvis-à-vis parental LNCaP cells (FIG. 7A). FIG. 7B depicts thedifferential protective effects of ectopic Bcl-xL on TG- andΔ2-TG-induced apoptotic death among the three Bcl-xL clones, in whichthe extent of cytoprotection correlated with the Bcl-xL expressionlevels. Overexpression of ectopic Bcl-xL conferred partial protection tothe cytotoxic effects of TG and Δ2-TG in B11 and B1 cells (P<0.01),whereas the excessive expression in B3 cells completely overcame theinhibitory effect of TG and Δ2-TG on Bcl-xL functions (P<0.01). Thisprotective effect was correlated with the inhibition of TG- andΔ2-TG-induced cytochrome c release (FIG. 7C; P<0.01).

Development of potent Δ2-TG-derived Bcl-xL/Bcl-2 binding inhibitors. TGhas previously been demonstrated to be effective in suppressing PC-3xenograft tumor growth at 500 g/kg/day (33). Dissociation of the effectof TG on apoptosis from PPARγ activation provided a molecular rationaleto structurally optimize Δ2-TG to develop potent Bcl-xL/Bcl-2 bindinginhibitors. Accordingly, we synthesized a series of Δ2-TG derivatives,and their activities in inhibiting BH3 domain-mediated Bcl-xL-Bakpeptide binding were examined (Chen, manuscript in preparation). Amongmore than 30 derivatives examined, TG-88 represented an optimal agentwith an-order-of-magnitude higher potency than Δ2-TG in FP-based Bcl-xLbinding inhibition (IC₅₀, 1.8±0.2 μM) and PC-3 cell proliferation (IC₅₀,2.5±0.2 μM). In addition, like Δ2-TG, TG-88 lacked appreciable activityin PPARγ activation. To examine its therapeutic relevance, we assessedthe in vivo effect of daily oral TG-88 at two different doses, 100 and200 mg/kg, on the growth of PC-3 xenograft tumors (FIG. 8). All animalstolerated the treatments well without observable signs of toxicity andwere characterized by stable body weights throughout the course ofstudy. No gross pathological abnormalities were noted at necropsy after63 days of treatment. As shown, both treatments displayed a significantinhibitory effect (P<0.05) when the tumors of control animals entered anexponential growth phase at day 35 and beyond. After 63 days oftreatment, the extents of tumor growth inhibition were 50% and 61% forgroups receiving 100 and 200 mg/kg/day, respectively.

Although accumulating evidence suggests that TG and CG mediatePPARγ-independent antitumor effects, the underlying mechanism remainsundefined. Here, we obtained several lines of evidence that the effectsof these TZDs on apoptosis in prostate cancer cells were attributable,in part, to the inhibition of Bcl-xL/Bcl-2 functions independently ofPPARγ activation. First, Δ2-TG and Δ2-CG, though devoid of PPARγactivity, exhibited slightly higher potency than TG and CG,respectively, in inducing apoptotic death irrespective of differences inPPARγ expression levels between LNCaP and PC-3 cells. In contrast, RGand PG, two TZDs currently in clinical use for the treatment ofdiabetes, lacked appreciable effects on apoptosis despite their higherpotency in PPARγ activation than TG and CG. Second, a correlation existsbetween the potency in inhibiting BH3 peptide binding to Bcl-xL or Bcl-2and the effectiveness in inducing apoptosis in prostate cancer cells.For example, the inability of RG and PG to trigger apoptotic death wasreflected in their weak potency in displacing BH3 domain-mediatedinteractions. It is interesting that introduction of a double bondadjoining the terminal thiazolidine-2,4-dione ring in TG and CG enhancedthe Bcl-xL/Bcl-2 inhibitory activity, while abrogating the ability toactivate PPARγ. Presumably, this change in pharmacological profiles wasattributable to the structural rigidity surrounding the heterocyclicsystem as a result of the double bond introduction. Third, theimmunoprecipitation study indicates that the level of Bak associatedwith Bcl-2 and Bcl-xL was greatly reduced in TG- and Δ2-TG-treated cellsas compared to DMSO control. Disruption of the BH3 domain-mediatedinteractions-led to the liberation of proapoptotic Bcl-2 members, whichcaused cells to undergo apoptosis by facilitating cytochrome c releaseand caspase-9 activation. This premise was borne out by the ability ofZ-VAD-FMK to protect cells from TG- and Δ2-TG-induced apoptosis. Fourth,overexpression of Bcl-xL provided LNCaP cells protection against TG- andΔ2-TG-induced apoptosis.

Considering the pivotal role of Bcl-xL and Bcl-2 in regulatingmitochondrial integrity, this new mode of action provides a molecularframework to account for the PPARγ-independent effects of TZDs onapoptotic death in cancer cells. It is also noteworthy that TG, CG, andtheir Δ2-derivatives lack specificity in recognizing Bcl-xL and Bcl-2.This relaxed specificity might prove advantageous in light of theimportance of both Bcl-2 members in regulating apoptosis thresholds tochemotherapeutic agents.

In summary, the impetus of the dissociation of the in vitro antitumoractivities of TZDs from PPARγ activation is multifold. First, althoughTG has been shown to reduce the growth of xenograft tumors in nude mice(33), this PPARγ agonist has also been reported to promote thedevelopment of colon tumors and enhance colon polyp formation inAPC^(Min) mice that are genetically predisposed to intestinal neoplasia(35, 36). Thus, a crucial issue that warrants investigation is the roleof PPARγ activation in tumorigenic promotion vis-à-vis antitumor effectsin these animal model studies. Conceivably, TZDs and theirPPARγ-inactive derivatives provide useful tools to shed light onto thelink between PPARγ activation and increased cancer risk. Second, from atranslational perspective, separation of these two pharmacologicalactivities provides molecular underpinnings to use TZDs, especiallyΔ2-TG and Δ2-CG, as molecular platforms to design BC1-xL/Bcl-2inhibitors with greater in vitro and in vivo antitumor potency. Third,due to the heterogeneous nature of prostate cancer, different prostatetumor cell lines display differential sensitivity to various apoptoticsignals. For example, PC-3 cells are able to resist apoptotic signalsemanating from withdrawal of trophic factors, and exposure to cytokinesand chemotherapeutic agents, in part, due to elevated levels of Aktactivation and Bcl-xL overexpression. Consequently, these molecules havetranslational relevance to be developed into antitumor agents for theprevention and/or therapy of cancers alone or in combination with othertreatments. The proof of principle for this premise was TG-88, a closestructural analogue of Δ2-TG, with an order-of-magnitude higher potencythan Δ2-TG in blocking Bcl-xL binding and inhibiting PC-3 cellproliferation. Oral TG-88 at 100 and 200 mg/kg/day was effective insuppressing PC-3 xenograft tumor growth without causing weight loss orapparent toxicity, indicating its oral bioavailability and potentialclinical use. Further development of these novel agents for theprevention and/or treatment of prostate cancer is currently underway.

The examples herein are for illustrative purposes only and are not meantto limit the scope of the invention.

REFERENCES

-   1. Day, C. Thiazolidinediones: a new class of antidiabetic drugs.    Diabet Med, 16: 179-192, 1999.-   2. Koeffler, H. P. Peroxisome proliferator-activated receptor gamma    and cancers. Clin Cancer Res, 9: 1-9, 2003.-   3. Tontonoz, P., Singer, S., Forman, B. M., Sarraf, P., Fletcher, J.    A., Fletcher, C. D., Brun, R. P., Mueller, E., Aitiok, S.,    Oppenheim, H., Evans, R. M., and Spiegelman, B. M. Terminal    differentiation of human liposarcoma cells induced by ligands for    peroxisome proliferator-activated receptor gamma and the retinoid X    receptor. Proc Natl Acad Sci USA, 94: 237-241, 1997.-   4. Gupta, R. A., Brockman, J. A., Sarraf, P., Willson, T. M., and    DuBois, R. N. Target genes of peroxisome proliferator-activated    receptor gamma in colorectal cancer cells. J Biol Chem, 276:    29681-29687, 2001.-   5. Altiok, S., Xu, M., and Spiegelman, B. M. PPARgamma induces cell    cycle withdrawal: inhibition of E2F/DP DNA-binding activity via    down-regulation of PP2A. Genes Dev, 11: 1987-1998, 1997.-   6. Palakurthi, S. S., Aktas, H., Grubissich, L. M., Mortensen, R.    M., and Halperin, J. A. Anticancer effects of thiazolidinediones are    independent of peroxisome proliferator-activated receptor gamma and    mediated by inhibition of translation initiation. Cancer Res, 61:    6213-6218, 2001.-   7. Takeda, K., Ichiki, T., Tokunou, T., lino, N., and Takeshita, A.    15-Deoxy-delta 12,14-prostaglandin J2 and thiazolidinediones    activate the MEK/ERK pathway through phosphatidylinositol 3-kinase    in vascular smooth muscle cells. J Biol Chem, 276: 48950-48955,    2001.-   8. Gourii-Berthold, I., Berthold, H. K., Weber, A. A., Ko, Y., Seul,    C., Vetter, H., and Sachinidis, A. Troglitazone and rosiglitazone    induce apoptosis of vascular smooth muscle cells through an    extracellular signal-regulated kinase-independent pathway. Naunyn    Schmiedebergs Arch Pharmacol, 363: 215-221, 2001.-   9. Bae, M. A. and Song, B. J. Critical role of c-Jun N-terminal    protein kinase activation in troglitazone-induced apoptosis of human    HepG2 hepatoma cells. Mol Pharmacol, 63: 401-408, 2003.-   10. Baek, S. J., Wilson, L. C., Hsi, L. C., and Eling, T. E.    Troglitazone, a peroxisome proliferator-activated receptor gamma    (PPAR gamma) ligand, selectively induces the early growth response-1    gene independently of PPAR gamma. A novel mechanism for its    anti-tumorigenic activity. J Biol Chem., 278: 5845-5853, 2003.-   11. Motomura, W., Okumura, T., Takahashi, N., Obara, T., and    Kohgo, Y. Activation of peroxisome proliferator-activated receptor    gamma by troglitazone inhibits cell growth through the increase of    p27KiP1 in human. Pancreatic carcinoma cells. Cancer Res, 60:    5558-5564, 2000.-   12. Sugimura, A., Kiriyama, Y., Nochi, H., Tsuchiya, H., Tamoto, K.,    Sakurada, Y., Ui, M., and Tokumitsu, Y. Troglitazone suppresses cell    growth of myeloid leukemia cell lines by induction of p21WAF1/CIP1    cyclin-dependent kinase inhibitor. Biochem Biophys Res Commun, 261:    833-837, 1999.-   13. Okura, T., Nakamura, M., Takata, Y., Watanabe, S., Kitami, Y.,    and Hiwada, K. Troglitazone induces apoptosis via the p53 and Gadd45    pathway in vascular smooth muscle cells. Eur J Pharmacol, 407:    227-235, 2000.-   14. Elstner, E., Muller, C., Koshizuka, K., Williamson, E. A., Park,    D., Asou, H., Shintaku, P., Said, J. W., Heber, D., and    Koeffler, H. P. Ligands for peroxisome proliferator-activated    receptorgamma and retinoic acid receptor inhibit growth and induce    apoptosis of human breast cancer cells in vitro and in BNX mice.    Proc Natl Acad Sci USA, 95: 8806-8811, 1998.-   15. Mueller, E., Smith, M., Sarraf, P., Kroll, T., Aiyer, A.,    Kaufman, D. S., Oh, W., Demetri, G., Figg, W. D., Zhou, X. P., Eng,    C., Spiegelman, B. M., and Kantoff, P. W. Effects of ligand    activation of peroxisome proliferator-activated receptor gamma in    human prostate cancer. Proc Natl Acad Sci USA, 97: 10990-10995,    2000.-   16. Yang, C. C., Lin, H. P., Chen, C. S., Yang, Y. T., Tseng, P. H.,    and Rangnekar, V. M. Bcl-xL mediates a survival mechanism    independent of the phosphoinositide 3-kinase/Akt pathway in prostate    cancer cells. J Biol Chem, 278: 25872-25878, 2003.-   17. Dandliker, W. B., Hsu, M. L., Levin, J., and Rao, B. R.    Equilibrium and kinetic inhibition assays based upon fluorescence    polarization. Methods Enzymol, 74 Pt C: 3-28, 1981.-   18. Chou, T. C. and Talalay, P. Quantitative analysis of dose-effect    relationships: the combined effects of multiple drugs or enzyme    inhibitors. Adv Enzyme Regul, 22: 27-55, 1984.-   19. Forman, B. M., Tontonoz, P., Chen, J., Brun, R. P.,    Spiegelman, B. M., and Evans, R. M. 15-Deoxy-delta 12,    14-prostaglandin 32 is a ligand for the adipocyte determination    factor PPAR gamma. Cell, 83: 803-812, 1995.-   20. Wang, J. L., Liu, D., Zhang, Z. J., Shan, S., Han, X.,    Srinivasula, S. M., Croce, C. M., Alnemri, E. S., and Huang, Z.    Structure-based discovery of an organic compound that binds Bcl-2    protein and induces apoptosis of tumor cells. Proc Natl Acad Sci    USA, 97: 7124-7129, 2000.-   21. Degterev, A., Lugovskoy, A., Cardone, M., Mulley, B., Wagner,    G., Mitchison, T., and Yuan, 3. Identification of small-molecule    inhibitors of interaction between the BH3 domain and Bcl-xL. Nat    Cell Biol, 3: 173-182, 2001.-   22. Tzung, S. P., Kim, K. M., Basanez, G., Giedt, C. D., Simon, J.,    Zimmerberg, J., Zhang, K. Y., and Hockenbery, D. M. Antimycin A    mimics a cell-death-inducing Bcl-2 homology domain 3. Nat Cell Biol,    3: 183-191, 2001.-   23. Enyedy, I. J., Ling, Y., Nacro, K., Tomita, Y., Wu, X., Cao, Y.,    Guo, R., Li, B., Zhu, X. Huang, Y., Long, Y. Q., Roller, P. P.,    Yang, D., and Wang, S. Discovery of small-molecule inhibitors of    Bcl-2 through structure-based computer screening. J Med Chem, 44:    4313-4324, 2001.-   24. Lugovskoy, A. A., Degterev, A. I., Fahmy, A. F., Zhou, P.,    Gross, J. D., Yuan, J., and Wagner, G. A novel approach for    characterizing protein ligand complexes: molecular basis for    specificity of small-molecule Bcl-2 inhibitors. J Am Chem Soc, 124:    1234-1240, 2002.-   25. Chan, S. L., Lee, M. C., Tan, K. O., Yang, L. K., Lee, A. S.,    Flotow, H., Fu, N.Y., Butler, M. S., Soejarto, D. D., Buss, A. D.,    and Yu, V. C. Identification of chelerythrine as an inhibitor of    BclXL function. J Biol Chem, 278: 20453-20456, 2003.-   26. Zhang, M., Liu, H., Guo, R., Ling, Y., Wu, X., Li, B.,    Roller, P. P., Wang, S., and Yang, D. Molecular mechanism of    gossypol-induced cell growth inhibition and cell death of HT-29    human colon carcinoma cells. Biochem Pharmacol, 66: 93-103, 2003.-   27. Cory, S., Huang, D. C., and Adams, J. M. The Bcl-2 family: roles    in cell survival and oncogenesis. Oncogene, 22: 8590-8607, 2003.-   28. Sattler, M., Liang, H., Nettesheim, D., Meadows, R. P.,    Harlan, J. E., Eberstadt, M., Yoon, H. S., Shuker, S. B., Chang, B.    S., Minn, A. J., Thompson, C. B., and Fesik, S. W. Structure of    Bcl-xL-Bak peptide complex: recognition between regulators of    apoptosis. Science, 275: 983-986, 1997.-   29. Diaz, J. L., Oltersdorf, T., Home, W., McConnell, M., Wilson,    G., Weeks, S., Garcia, T., and Fritz, L. C. A common binding site    mediates heterodimerization and homodimerization of Bcl-2 family    members. J Biol Chem, 272: 11350-11355, 1997.-   30. Otter, I., Conus, S., Ravn, U., Rager, M., Olivier, R., Monney,    L., Fabbro, D., and Borner, C. The binding properties and biological    activities of Bcl-2 and Bax in cells exposed to apoptotic stimuli. J    Biol Chem, 273: 6110-6120, 1998.-   31. Nouraini, S., Six, E., Matsuyama, S., Krajewski, S., and    Reed, J. C. The putative pore-forming domain of Bax regulates    mitochondrial localization and interaction with Bcl-X(L). Mol Cell    Biol, 20: 1604-1615, 2000.-   32. Finnegan, N. M., Curtin, J. F., Prevost, G., Morgan, B., and    Cotter, T. G. Induction of apoptosis in prostate carcinoma cells by    BH3 peptides which inhibit Bak/Bcl-2 interactions. Br J Cancer, 85:    115-121, 2001.-   33. Kubota, T., Koshizuka, K., Williamson, E. A., Asou, H., Said, J.    W., Holden, S., Miyoshi, I., and Koeffler, H. P. Ligand for    peroxisome proliferator-activated receptor gamma (troglitazone) has    potent antitumor effect against human prostate cancer both in vitro    and in vivo. Cancer Res, 58: 3344-3352, 1998.-   34. Sarraf, P., Mueller, E., Jones, D., King, F. J., DeAngelo, D.    J., Partridge, J. B., Holden, S. A., Chen, L. B., Singer, S.,    Fletcher, C., and Spiegelman, B. M. Differentiation and reversal of    malignant changes in colon cancer through PPARgamma. Nat Med, 4:    1046-1052, 1998.-   35. Lefebvre, A. M., Chen, I., Desreumaux, P., Najib, J.,    Fruchart, J. C., Geboes, K., Briggs, M., Heyman, R., and Auwerx, J.    Activation of the peroxisome proliferator-activated receptor gamma    promotes the development of colon tumors in C57BL/6J-APCMin/+ mice.    Nat Med, 4: 1053-1057, 1998.-   36. Saez, E., Tontonoz, P., Nelson, M. C., Alvarez, J. G., Ming, U.    T., Baird, S. M., Thomazy, V. A., and Evans, R. M. Activators of the    nuclear receptor PPARgamma enhance colon polyp formation. Nat Med,    4: 1058-1061, 1998.

1-18. (canceled)
 19. A method of treating prostate cancer in a subjectpreviously identified as having an unwanted cell proliferation of theprostate, the process comprising administering to the subject atherapeutically effective amount of at least one compound of thefollowing formula I, formula III, formula IV, formula V, formula VI,formula VII, formula VIII, formula X or formula XI:

wherein R is selected from aryl, heteroaryl, cycloalkyl,heterocycloalkyl, alkylaryl, and combinations thereof; and wherein R maybe substituted at one or more substitutable positions with one or moresubstituents selected from the group consisting of hydroxyl, alkyl, andcombinations thereof;

wherein X₁ is H, alkyl, alkoxy, halo, nitro, haloalkylaryl, haloaryl,alkylaryl, and combinations thereof; and X₂ is alkyl other than methyl,alkoxy other than methyl, halo other than bromo, and combinationsthereof;

wherein X₁ is selected from H and halo; and Y is selected fromalkylaryl, ankenylaryl, alkenyl, ester carboxylic acids, ester alcohols,and combinations thereof;

wherein X₁ is selected from H and halo; and Y is selected fromstraight-chain alkenyl, branched alkenyl, and combinations thereof;

wherein X₁ is selected from H, alkoxy, halo, and combinations thereof;and Z is selected from

wherein X₁ is selected from H and halo, and Z is selected from N

wherein W is selected from O and S; Y is selected from straight chainalkenyl, branched alkenyl and combinations thereof; and Z′ is carboxylicacid;

wherein Y is selected from straight chain alkenyl, branched alkenyl andcombinations thereof; and Z′ is carboxylic acid;

wherein Y is selected from straight chain alkenyl, branched alkenyl andcombinations thereof; and Z′ is carboxylic acid; and pharmaceuticallyacceptable salts thereof.
 20. The method according to claim 19, whereinthe compound is administered orally, intravenously, intramuscularly,subcutaneously, or intraperitoneally.
 21. The method according to claim20, wherein the therapeutically effective amount administered is in therange of about 0.1-2000 mg.
 22. The method according to claim 20,wherein the therapeutically effective amount administered is in therange of about 1-200 mg per kg of body weight of the subject.
 23. Themethod of claim 19, wherein a compound of formula I is administered. 24.The method of claim 23, wherein R is selected from


25. The method of claim 19, wherein a compound of formula IV isadministered.
 26. The method of claim 25, wherein X₁ is selected from Hand Br.
 27. The method of claim 26, wherein R is selected from


28. The method of claim 19, wherein a compound of formula V isadministered.
 29. The method of claim 28, wherein X₁ is selected from Hand Br.
 30. The method of claim 29, wherein Y is selected from


31. The method of claim 19, wherein a compound of formula VI isadministered.
 32. The method of claim 31, wherein X₁ is selected from H,methoxy and ethoxy.
 33. The method of claim 32, wherein Z is selectedfrom


34. The method of claim 19, wherein a compound of formula VII isadministered.
 35. The method of claim 34, wherein X₁ is selected from H,Br and Cl.
 36. The method of claim 35, wherein Z is selected from


37. The method of claim 19, wherein a compound of formula VIII isadministered.
 38. The method of claim 37, wherein Y is


39. The method of claim 19, wherein a compound of formula X isadministered.
 40. The method of claim 39, wherein Y is


41. The method of claim 19, wherein a compound of formula XI isadministered.
 42. The method of claim 39, wherein Y is